The present invention relates to compositions comprising bacterial extracts useful for treating medical conditions such as respiratory disorders. The extracts may comprise bacterial lysates from cultures chosen from the following species:                Moraxella (Branhamella) catarrhalis, Moraxella (Moraxella) catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguinis, Staphylococcus Hemolyticus, Enterococcus faecalis, Streptococcus mutans, Streptococcus anginosus, Streptococcus mitis, Streptococcus salivarius (aka. Streptococcus viridans), Neisseria sicca, Hemophilus parainfluenzae, Actinobacillus (Hemophilus) actinomycetemcomitans, and Eikenella corrodens.         
In some embodiments, the extracts comprise at least one strain from each of the above species of bacteria, while in other embodiments, one or more specific strains from the list above may be removed or substituted with one or more different strains. Some embodiments of the present invention comprise an extract obtained from each of the following bacterial strains: Moraxella (Branhamella) catarrhalis 3622, Moraxella (Branhamella) catarrhalis 3625, Moraxella (Branhamella) catarrhalis I-045, Haemophilus influenzae 8467, Klebsiella pneumoniae ssp. ozaenae 5050, Klebsiella pneumoniae 204, Klebsiella pneumoniae 5056, Staphylococcus aureus I-049, Staphylococcus aureus I-050, Staphylococcus aureus I-051, Staphylococcus aureus I-052, Staphylococcus aureus I-053, Staphylococcus aureus I-054, Streptococcus (Diplococcus) pneumoniae 7465, Streptococcus (Diplococcus) pneumoniae 7466, Streptococcus (Diplococcus) pneumoniae 7978, Streptococcus (Diplococcus) pneumoniae 10319, Streptococcus pyogenes 8191, Streptococcus sanguinis I-046, Streptococcus sanguinis I-047, Streptococcus sanguinis I-048, Staphylococcus Hemolyticus 11042, Enterococcus faecalis 103015, Streptococcus mutans 10449, Streptococcus anginosus 10713, Streptococcus mitis 12261, Streptococcus salivarius 102503, Neisseria sicca 103345, Haemophilus parainfluenzae 7857, Actinobacillus (Hemophilus) actinomycetemcomitans 52.105, and Eikenella corrodens 10596. Those strains are deposited according to the Budapest Treaty. The strains indicated in the list with I-number were indexed by the Collection Nationale de Culture des Microorganismes at the Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France. All of the other strains were indexed by the National Collection of Type Cultures in London.
In some embodiments, one or more of the specific strains listed above may be omitted, or substituted with a different strain from the same species or from a different species of bacteria. For example, in some embodiments, one or more or even all of strains Staphylococcus Hemolyticus 11042, Enterococcus faecalis 103015, Streptococcus mutans 10449, Streptococcus anginosus 10713, Streptococcus mitis 12261, Streptococcus salivarius 102503, Neisseria sicca 103345, Haemophilus parainfluenzae 7857, Actinobacillus (Hemophilus) actinomycetemcomitans 52.105, and Eikenella corrodens 10596 may be omitted. In other embodiments, one or more Moraxella, Klebsiella, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus sanguinis strains may be omitted. Further, to aid digestion, a Lactobacillus strain or another strain of bacteria may also be used.
The extracts may be obtained by a process of alkaline lysis after cells are grown to a suitable optical density in a culture medium. In some embodiments, the bacteria are each grown on a medium that does not pose a risk of prion-related diseases or a risk of other diseases that may be transmitted through ingesting products obtained from animal-based media. For example, in some embodiments a vegetable-based medium is used to grow the cells, such as a soya-based medium. In other embodiments, a synthetic medium is used for cell growth. In yet other embodiments, a medium may include biological extracts such as yeast extract and horse serum, which also do not pose such disease risks.
The lysates may also be filtered to remove nucleic acids and larger cellular debris. In consequence of the filtration, in some embodiments, the amount of nucleic acid present in the extracts is less than 100 μg/mL. In some embodiments, insolubilized compounds such as cell wall debris and insufficiently degraded lipopolysaccharide (LPS) are also removed by the filtration. Hence, in some embodiments, the resulting extract comprises soluble molecular components and does not contain significant amounts of insoluble or particulate material.
Saccharide components may be preserved in the extracts, including lipopolysaccharide (LPS) components. During the lysis process, saccharides may become chemically modified, for example, cleaved into smaller structures or substituted with other functional groups.
Racemization of amino acids during the lysis process also creates D-amino acids from the naturally occurring L-amino acids found in natural proteins. D-amino acids can be beneficial in increasing bioavailability of the extracts, as proteins constituted principally or partially from D-amino acids are not efficiently digested in the mammalian gut. Thus, antigenic molecules in the extracts that are chemically modified during lysis to contain D-amino acids remain in the patient's body for a longer time, allowing potentially for a stronger immunostimulating action.
While bacterial extracts have been used in the prior art to stimulate the immune system against respiratory diseases, there has been a need to better standardize and control those extracts in order to make them safer, more effective, and longer lasting. For instance, it was previously thought that saccharide components, including potentially toxic lipopolysaccharide (LPS) components should be removed from bacterial extracts for safety reasons. (See, e.g., U.S. Pat. No. 5,424,287.) However, the instant invention provides a process that results in sufficient chemical modifications of LPS components that saccharides be safely retained. Retaining those components may improve efficacy and provide additional antigens to the extracts.
For example, the inventors have discovered that adjusting the pH and the time of lysis may allow for sufficient degradation of potentially allergenic or toxic cell wall components. Prior lysis conditions at lower pH's or shorter times, in contrast, produced extracts in which cell wall components and saccharides were insufficiently chemically modified. (See, e.g., GB 2 021 415 A.) The resulting extracts were too allergenic to be safely administered to patients. In general, the inventors have discovered that products lysed at too low a pH and/or at too short a time had higher toxicity, lower protein extraction, and lower filterability.
The filtration process may also influence the properties of the resulting extract, as the pore size of the filter, and in some cases, the chemical properties of the filter surface, may alter the type of materials that were removed and retained. For example, some embodiments of the instant invention use a filtration process designed to retain saccharides but to remove other molecular components such as nucleic acids.
Thus, the instant invention provides parameters that standardize the bacterial extracts to help maintain consistent safety and efficacy.